Abstract
Insulin receptor substrate-1 (IRS-1) is phosphorylated on multiple tyrosine residues by ligand-activated insulin receptors. These tyrosine phosphorylation sites serve to dock several Src homology 2-containing signaling proteins. In addition, IRS-1 contains a pleckstrin homology domain and a phosphotyrosine binding domain (PTB) implicated in protein-protein and protein-lipid interactions. In a yeast two-hybrid screening using Xenopus IRS-1 (xIRS-1) pleckstrin homology-PTB domains as bait, we identified a Xenopus homolog of Rho-associated kinase alpha (xROKalpha) as a potential xIRS-1-binding protein. The original clone contained the carboxyl terminus of xROKalpha (xROK-C) including the putative Rho binding domain but lacking the amino-terminal kinase domain. Further analyses in yeast indicated that xROK-C bound to the putative PTB domain of xIRS-1. Binding of xROK-C to xIRS-1 was confirmed in Xenopus oocytes after microinjection of mRNA corresponding to xROK-C. Furthermore, microinjection of xROK-C mRNA inhibited insulin-induced mitogen-activated protein kinase activation with a concomitant inhibition of oocyte maturation. In contrast, microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral Ras (v-Ras) mRNA. These results suggest that xROKalpha may play a role in insulin signaling via a direct interaction with xIRS-1.
Highlights
Phosphorylation of insulin receptor substrate-1 (IRS-1)1 by ligand-activated insulin receptors serves to dock several Src homology 2 domain-containing proteins [1,2,3,4]
Microinjection of xROK-C mRNA did not inhibit mitogen-activated protein kinase activation or oocyte maturation induced by progesterone or by microinjection of viral Ras (v-Ras) mRNA. These results suggest that xROK␣ may play a role in insulin signaling via a direct interaction with Xenopus IRS-1 (xIRS-1)
We report here that the Xenopus homolog of RhoA-associated protein kinase is an xIRS-1-binding protein and that binding of a noncatalytic region of xROK␣ to endogenous xIRS-1 correlates with inhibition of insulin signaling in Xenopus oocytes
Summary
Phosphorylation of insulin receptor substrate-1 (IRS-1) by ligand-activated insulin receptors serves to dock several Src homology 2 domain-containing proteins [1,2,3,4]. Biochemical and structural studies indicate that amino acids 161–265 of rat IRS-1 include the required component of the PTB domain which binds the NPEpY motif of the insulin receptor [9, 10]. Using the yeast two-hybrid assay, Gustafson and colleagues have provided evidence that additional amino acids that are carboxyl-terminal to the PTB domain (termed the SAIN domain) are necessary for binding to the NPEpY sequence of the insulin receptor [15, 16]. The putative PH and PTB domains of xIRS-1 are highly similar to those of rat IRS-1, with 85 and 90% amino acid sequence identity, respectively. To identify novel proteins that may bind the highly conserved amino-terminal protein modules (PH and PTB in particular) of IRS-1, we conducted a yeast two-hybrid screening of a Xenopus oocyte cDNA library. We report here that the Xenopus homolog of RhoA-associated protein kinase (xROK␣) is an xIRS-1-binding protein and that binding of a noncatalytic region of xROK␣ to endogenous xIRS-1 correlates with inhibition of insulin signaling in Xenopus oocytes
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